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Cell-fractionation analysis of glucan synthase I and II distribution and polysaccharide secretion in soybean protoplasts

Evidence for the involvement of coated vesicles in wall biogenesis

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Abstract

The organelles of soybean (Glycine max (L.) Merr.) protoplasts were separated using a recently developed procedure which allows rapid (3-h) recovery of a fraction enriched for coated vesicles (CVs). As determined by marker-enzyme enrichment and ultrastructural analysis of isolated membrane fractions, endoplasmic reticulum, Golgi membranes, glucan-synthase-II (EC 2.4.1.34)-containing membranes (putative plasma membrane), mitochondria, and CVs were enriched in separate fractions in a sucrose density gradient. Glucan synthase I (EC 2.4.1.12) had the highest specific activity in the Golgi-enriched and CV-enriched fractions and was found to comigrate with CVs upon rate-zonal centrifugation of a CV-enriched fraction. For further elucidation of the role of these latter organelles in cell-wall regeneration, freshly isolated protoplasts were pulsed with [3H]glucose for 20 min, and the disappearance of label from the organelles was followed for the ensuing 1 h. Although a CV-enriched fraction contained glucan synthase I, it contained very small amounts of labelled polysaccharide during the period of study. Pulse-chase experiments with [3H]glucose helped to confirm the role of the Golgi apparatus in secretion of matrix polysaccharides by protoplasts.

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Abbreviations

CV(s):

coated vesicle(s)

Da:

dalton

ER:

endoplasmic reticulum

GSI,II:

glucan synthase I and II, respecitively

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Authors and Affiliations

  1. Biology Department, University of Saskatchewan, S7N 0W0, Saskatoon, SK, Canada

    L. R. Griffing & L. C. Fowke

  2. Crop Science Department, University of Guelph, N1G 2W1, Guelph, ONT, Canada

    B. G. Mersey

Authors
  1. L. R. Griffing
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  2. B. G. Mersey
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  3. L. C. Fowke
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Two whom correspondence should be directed. Address after February 1986:Department of Biology, Texas A&M University. College Station, TX 77843-3258, USA

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Griffing, L.R., Mersey, B.G. & Fowke, L.C. Cell-fractionation analysis of glucan synthase I and II distribution and polysaccharide secretion in soybean protoplasts. Planta 167, 175–182 (1986). https://doi.org/10.1007/BF00391412

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  • DOI: https://doi.org/10.1007/BF00391412

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